COVID-19, Coenzyme Q10 and Selenium.

In COVID-19 infection, a balance must be achieved in immune defence against the virus without precipitating a cytokine storm, which is responsible for lung injury and respiratory distress in severe cases. The initial immune response and the subsequent resolution of inflammation are likely to be dependent on nutritional status, as one contributing factor. Here, we have reviewed the potential link between two specific nutrients, coenzyme Q10 (CoQ10) and selenium, with effects on oxidative stress and inflammation in viral infection. We conclude that both reagents show promise in the treatment of patients with COVID-19 disease. This could give particular relevance over the next several months as promising vaccines are deployed to minimise the COVID-19 spread and as a potential preventative or mitigating approach for future epidemics and pandemics.

Supplementation with selenium and coenzyme Q10 in critically ill patients.

Multiple organ dysfunction and resultant mortality in critically ill patients has been linked with impaired cellular energy supply and oxidative stress. Clinical studies supplementing selenium, on the basis of its role as a key cofactor of antioxidant enzymes, have reported variable outcomes in critically ill patients. However, the synergistic interaction between selenium and coenzyme Q10, which has essential roles in cellular energy supply and as an antioxidant, has not been considered in such studies. This article reviews the link between selenium and coenzyme Q10, and the potential role of their co-supplementation in critical illness.

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'.

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.

Successful reversal of propionic acidaemia associated cardiomyopathy: evidence for low myocardial coenzyme Q10 status and secondary mitochondrial dysfunction as an underlying pathophysiological mechanism.

Dilated cardiomyopathy is a rare complication in propionic acidaemia (PA). Underlying pathophysiological mechanisms are poorly understood. We present a child of Pakistani consanguineous parents, diagnosed with late-onset PA at 18months of age. He presented a mild phenotype, showed no severe further decompensations, normal growth and psychomotor development on a low protein diet and carnitine supplementation. At 15years, a mildly dilated left ventricle was noticed. At 17years he presented after a 2-3month history of lethargy and weight loss with severe decompensated dilated cardiomyopathy. He was stabilised on inotropic support and continuous haemofiltration; a Berlin Heart biventricular assist device was implanted. He received d,l-hydroxybutyrate 200mg/kg/day, riboflavin and thiamine 200mg/day each and coenzyme Q10 (CoQ10). Myocardial biopsy showed endocardial fibrosis, enlarged mitochondria, with atypical cristae and slightly low respiratory chain (RC) complex IV activity relative to citrate synthase (0.012, reference range 0.014-0.034). Myocardial CoQ10 was markedly decreased (224pmol/mg, reference range 942-2738), with a marginally decreased white blood cell level (34pmol/mg reference range 37-133). The dose of CoQ10 was increased from 1.5 to 25mg/kg/day. Cardiomyopathy slowly improved allowing removal of the external mechanical cardiac support after 67days. We demonstrate for the first time low myocardial CoQ10 in cardiomyopathy in PA, highlighting secondary mitochondrial impairment as a relevant causative mechanism. According to these findings, a high-dose CoQ10 supplementation could be a potential adjuvant therapeutic to be considered in PA-related cardiomyopathy.

Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Coenzyme Q10 as a therapy for mitochondrial disease.

Treatment of mitochondrial respiratory chain (MRC) disorders is extremely difficult, however, coenzyme Q10 (CoQ10) and its synthetic analogues are the only agents which have shown some therapeutic benefit to patients. CoQ10 serves as an electron carrier in the MRC as well as functioning as a potent lipid soluble antioxidant. CoQ10 supplementation is fundamental to the treatment of patients with primary defects in the CoQ10 biosynthetic pathway. The efficacy of CoQ10 and its analogues in the treatment of patients with MRC disorders not associated with a CoQ10 deficiency indicates their ability to restore electron flow in the MRC and/or increase mitochondrial antioxidant capacity may also be important contributory factors to their therapeutic potential.

Effect of Coenzyme Q10 supplementation on mitochondrial electron transport chain activity and mitochondrial oxidative stress in Coenzyme Q10 deficient human neuronal cells.

Primary Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with a heterogeneous clinical presentation. Common presenting features include both muscle and neurological dysfunction. Muscle abnormalities can improve, both clinically and biochemically following CoQ10 supplementation, however neurological symptoms are only partially ameliorated. At present, the reasons for the refractory nature of the neurological dysfunction remain unknown. In order to investigate this at the biochemical level we evaluated the effect of CoQ10 treatment upon a previously established neuronal cell model of CoQ10 deficiency. This model was established by treatment of human SH-SY5Y neuronal cells with 1 mM para-aminobenzoic acid (PABA) which induced a 54% decrease in cellular CoQ10 status. CoQ10 treatment (2.5 µM) for 5 days significantly (p<0.0005) decreased the level of mitochondrial superoxide in the CoQ10 deficient neurons. In addition, CoQ10 treatment (5 µM) restored mitochondrial membrane potential to 90% of the control level. However, CoQ10 treatment (10 µM) was only partially effective at restoring mitochondrial electron transport chain (ETC) enzyme activities. ETC complexes II/III activity was significantly (p<0.05) increased to 82.5% of control levels. ETC complexes I and IV activities were restored to 71.1% and 77.7%, respectively of control levels. In conclusion, the results of this study have indicated that although mitochondrial oxidative stress can be attenuated in CoQ10 deficient neurons following CoQ10 supplementation, ETC enzyme activities appear partially refractory to treatment. Accordingly, treatment with >10 µM CoQ10 may be required to restore ETC enzyme activities to control level. Accordingly, these results have important implication for the treatment of the neurological presentations of CoQ10 deficiency and indicate that high doses of CoQ10 may be required to elicit therapeutic efficacy.

PGC-1ß mediates adaptive chemoresistance associated with mitochondrial DNA mutations.

Primary mitochondrial dysfunction commonly leads to failure in cellular adaptation to stress. Paradoxically, however, nonsynonymous mutations of mitochondrial DNA (mtDNA) are frequently found in cancer cells and may have a causal role in the development of resistance to genotoxic stress induced by common chemotherapeutic agents, such as cis-diammine-dichloroplatinum(II) (cisplatin, CDDP). Little is known about how these mutations arise and the associated mechanisms leading to chemoresistance. Here, we show that the development of adaptive chemoresistance in the A549 non-small-cell lung cancer cell line to CDDP is associated with the hetero- to homoplasmic shift of a nonsynonymous mutation in MT-ND2, encoding the mitochondrial Complex-I subunit ND2. The mutation resulted in a 50% reduction of the NADH:ubiquinone oxidoreductase activity of the complex, which was compensated by increased biogenesis of respiratory chain complexes. The compensatory mitochondrial biogenesis was most likely mediated by the nuclear co-activators peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) and PGC-1ß, both of which were significantly upregulated in the CDDP-resistant cells. Importantly, both transient and stable silencing of PGC-1ß re-established the sensitivity of these cells to CDDP-induced apoptosis. Remarkably, the PGC-1ß-mediated CDDP resistance was independent of the mitochondrial effects of the co-activator. Altogether, our results suggest that partial respiratory chain defects because of mtDNA mutations can lead to compensatory upregulation of nuclear transcriptional co-regulators, in turn mediating resistance to genotoxic stress.

Assessment of mitochondrial respiratory chain function in hyperphenylalaninaemia.

Phenylketonuria (PKU) is an autosomal recessive disorder resulting in neurological and intellectual disability when untreated. However, even in treated patients there may be residual neurological impairment such as tremor. It has been suggested that the hyperphenylalaninaemia in patients with PKU reduces complex I (NADH:ubiquinone reductase) activity of the mitochondrial respiratory chain (MRC) and/or biosynthesis of coenzyme Q(10) (CoQ(10)), which acts as an electron carrier in the MRC, leading to impaired energy metabolism in the brain of patients with PKU and hence the neurological pathology. The aim of this study was to elucidate the mechanism of phenylalanine (Phe) toxicity on the MRC. We compared mean plasma and blood-spot Phe and mononuclear CoQ(10) levels in 17 patients with PKU and a tremor compared to 22 patients without tremor. Human 1321N1 astrocytoma cells were exposed to hyperphenylalaninaemia by the addition of 300 or 900 micromol/L of Phe to the cell culture medium. Following 96 h of culture we measured complex I and citrate synthase activities and CoQ(10) level. Results showed no significant difference in Phe or CoQ(10) levels in patients with tremor compared to those without tremor. Further, hyperphenylalaninaemia did not cause a significant reduction in complex I activity or CoQ(10) biosynthesis, even when taking into account the mitochondrial enrichment of the cell samples by expressing complex I and CoQ(10) as a ratio to citrate synthase. In conclusion, the results of this study suggest that hyperphenylalaninaemia does not contribute to the pathophysiology of PKU by causing a decrease in MRC complex I activity and/or CoQ(10) biosynthesis.

Mitochondrial cytochrome c release: a factor to consider in mitochondrial disease?

The pathogenesis of mitochondrial disorders has largely focused on the impairment of cellular energy metabolism. However, mitochondrial dysfunction has also been implicated as a factor in the initiation of apoptosis due to the translocation of cytochrome c, from mitochondria to the cytosol, and the subsequent cleavage of pro-caspase 3. In this study, we determined the cytochrome c content of cytosols (skeletal muscle) prepared from 22 patients with evidence of compromised mitochondrial electron transport chain enzyme activity and 26 disease controls. The cytochrome c content of the mitochondrial electron transport chain-deficient group was found to be significantly (p < 0.02) elevated when compared with the control group (63.7 +/- 15.5 versus 27.7 +/- 2.5 ng/mg protein). Furthermore, a relationship between the cytosolic cytochrome c content of skeletal muscle and complex I and complex IV activities was demonstrated. Such data raise the possibility that mitochondrial cytochrome c release may be a feature of mitochondrial disorders, particularly for those patients with marked deficiencies of respiratory chain enzymes. Whether initiation of apoptosis occurs as a direct consequence of this cytochrome c release has not been fully evaluated here. However, for one patient with the greatest documented cytosolic cytochrome c content, caspase 3 could be demonstrated in the cytosolic preparation. Further work is required in order to establish whether a relationship also exists between caspase 3 formation and the magnitude of respiratory chain deficiency.

Inhibition of mitochondrial complex IV leads to secondary loss complex II-III activity: implications for the pathogenesis and treatment of mitochondrial encephalomyopathies.

Mitochondrial encephalomyopathies, arising from deficiencies of the electron transport chain (ETC) give rise to a wide clinical spectrum of presentation and are often progressive in nature. The aetiology of mitochondrial encephalomyopathies have yet to be fully elucidated, however, a successive loss of ETC function may contribute to the progressive nature of these disorders. The possibility arises that as a consequence of a primary impairment of ETC activity, secondary damage to the ETC may occur. In order to investigate this hypothesis, we established a model of cytochrome oxidase (Complex IV) deficiency in cultured human astrocytoma 1321N cells. Potassium cyanide (KCN, 1mM) resulted in a sustained 50% (p<0.01) loss of complex IV. At 24h activities of the other ETC complexes were unaffected. However, at 72h significant loss of succinate-cytochrome c reductase (complex II-III) activity expressed as a ratio to the mitochondrial marker, citrate synthase was observed. (KCN treated; 0.065+/-0.011 vs controls; 0.118+/-0.017 mean+/-SEM, n=8, p<0.05). These results provide a possible mechanism for the progressive nature of ETC defects and why in some patients multiple patterns of ETC deficiencies can be demonstrated.

PINK1 protein in normal human brain and Parkinson's disease.

Parkinson's disease is a common incurable neurodegenerative disease whose molecular aetiology remains unclear. The identification of Mendelian genes causing rare familial forms of Parkinson's disease has revealed novel proteins and pathways that are likely to be relevant in the pathogenesis of sporadic Parkinson's disease. Recently, mutations in a novel gene, PINK1, encoding a 581 amino acid protein with both mitochondrial targeting and serine/threonine kinase domains, were identified as a cause of autosomal recessive parkinsonism. This provided important evidence for the role of the mitochondrial dysfunction and kinase pathways in neurodegeneration. In this study, we report the first characterization of the PINK1 protein in normal human and sporadic Parkinson's brains, in addition to Parkinson's cases with heterozygous PINK1 mutations. The possible role of the PINK1 protein was also assessed in a number of neurodegenerative diseases characterized by proteinaceous inclusions. For these studies, rabbit polyclonal antibodies were raised against two peptide sequences within the N-terminal hydrophilic loops of PINK1 protein. Using immunohistochemistry and western blotting we were able to demonstrate that PINK1 is a ubiquitous protein expressed throughout the human brain and it is found in all cell types showing a punctate cytoplasmic staining pattern consistent with mitochondrial localization. Fractionation studies of human and rat brain confirm that PINK1 is localized to the mitochondrial membranes. In addition, we show that PINK1 is detected in a proportion of Lewy bodies in cases of sporadic Parkinson's disease and Parkinson's disease associated with heterozygous mutations in the PINK1 gene, which are clinically and pathologically indistinguishable from the sporadic cases. PINK1 was absent in cortical Lewy bodies, in neurofibrillary tangles in Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration, and in the glial and neuronal alpha-synuclein positive inclusions in multiple system atrophy. These studies provide for the first time in vivo morphological and biochemical evidence to support a mitochondrial localization of PINK1 and underpin the significance of mitochondrial dysfunction in the pathogenesis of nigral cell degeneration in Parkinson's disease.

The significance of reduced respiratory chain enzyme activities: clinical, biochemical and radiological associations.

BACKGROUND: Mitochondrial diseases are an important group of neurometabolic disorders in children with varied clinical presentations and diagnosis that can be difficult to confirm. AIM: To report the significance of reduced respiratory chain enzyme (RCE) activity in muscle biopsy samples from children. METHODS: Retrospective odds ratio was used to compare clinical and biochemical features, DNA studies, neuroimaging, and muscle biopsies in 18 children with and 48 without reduced RCE activity. RESULTS: Children with reduced RCE activity were significantly more likely to have consanguineous parents, to present with acute encephalopathy and lactic acidaemia and/or within the first year of life; to have an axonal neuropathy, CSF lactate >4 mmol/l; and/or to have signal change in the basal ganglia. There were positive associations with a maternal family history of possible mitochondrial cytopathy; a presentation with failure to thrive and lactic acidaemia, ragged red fibres, reduced fibroblast fatty acid oxidation and with an abnormal allopurinol loading test. There was no association with ophthalmic abnormalities, deafness, epilepsy or myopathy. CONCLUSION: The association of these clinical, biochemical and radiological features with reduced RCE activity suggests a possible causative link.

Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Glutathione deficiency in patients with mitochondrial disease: implications for pathogenesis and treatment.

Glutathione (GSH) is a key intracellular antioxidant. With regard to mitochondrial function, loss of GSH is associated with impairment of the electron transport chain (ETC). Since GSH biosynthesis is an energy-dependent process, we postulated that in patients with ETC defects GSH status becomes compromised, leading to further loss of ETC activity. We performed electrochemical HPLC analysis to determine the GSH concentration of 24 skeletal muscle biopsies from patients with defined ETC defects compared to 15 age-matched disease controls. Comparison of these groups revealed a significant (p < 0.001) decrease in GSH concentration in the ETC-deficient group: 7.7 +/- 0.9 vs 12.3 +/- 0.6 nmol/mg protein in the control group. Further analysis of the data revealed that patients with multiple defects of the ETC had the most marked GSH deficiency: 4.1 +/- 0.9 nmol/mg protein (n = 4, p < 0.05) when compared to the control group. These findings suggest that a deficiency in skeletal muscle GSH concentration is associated with an ETC defect, possibly as a consequence of diminished ATP availability or increased oxidative stress. The decreased ability to combat oxidative stress could therefore cause further loss of ETC activity and hence be a contributing factor in the progressive nature of this group of disorders. Furthermore, restoration of cellular GSH status could prove to be of therapeutic benefit in patients with a GSH deficiency associated with their ETC defects.

Chlorimipramine: a novel anticancer agent with a mitochondrial target.

Mitochondria have been suggested to be a potential intracellular target for cancer chemotherapy. In this report, we demonstrate the ability of the tricyclic antidepressant chlorimipramine to kill human glioma cells in vitro by a molecular mechanism resulting in an increase in caspase 3 activity following inhibition of glioma oxygen consumption. Studies with isolated rat mitochondria showed that chlorimipramine specifically inhibited mitochondrial complex III activity, which causes decreased mitochondrial membrane potential as well as mitochondrial swelling and vacuolation. The use of chlorimipramine in human as an effective, non-toxic cancer therapeutic having a strong selectivity between cancer cells and normal cells on the basis of their mitochondrial function is discussed.

Wrist arthrodesis using a Synthes wrist fusion plate.

Thirty-nine patients were retrospectively reviewed after a wrist arthrodesis using a Synthes wrist fusion plate and iliac crest bone graft. Information was obtained from review of patient files, a questionnaire to assess pain, function and work status, and clinical assessment of grip strength, forearm rotation and fingers motion. All wrist fusions united except that the index carpometacarpal joint failed to unite in one patient. Thirty-seven patients were satisfied with the procedure, noting a reduction in wrist pain after fusion, but all reported some limitation of function. The wrist fusion plate was removed in six patients and a further four patients experienced minor symptoms over the dorsal aspect of the middle finger metacarpal.

Primary pyruvate dehydrogenase E3 binding protein deficiency with mild hyperlactataemia and hyperalaninaemia.

A case of pyruvate dehydrogenase E3 binding protein deficiency is reported in a 24-year-old male with encephalomyopathy. Blood lactate was only minimally elevated, as was alanine.

N-acetylcysteine and Unverricht-Lundborg disease: variable response and possible side effects.

Serum glutathione levels were assessed in a patient with genetically proven Unverricht-Lundborg disease (ULD) before and during treatment with the antioxidant N-acetylcysteine (NAC). Glutathione levels were low before treatment, and increased during treatment. This increase was mirrored by an improvement in seizures, but not in myoclonus or ataxia. Three other patients with clinically determined ULD showed a variable response and some notable side effects during treatment with NAC.